1. Field of the Invention
The present invention relates to lipid coupling reagents, and in particular, to a reagent for coupling amine-containing molecules, such as protein molecules, to a lipid-surface structure, such as a liposome.
2. References
The following references are referred to herein by corresponding number:
1. Tyrell, D. A., Biochem Biophys Acta (1976) 457: 259. PA0 2. Dunnick, J. K., J Nucl Med (1975) 16: 483. PA0 3. Torchilin, V. P., et al, Biochem Biophys Res Commun (1979) 85: 1114. PA0 4. Torchilin, V. P., Biochem Biophys Res Commun (1978) 85: 983. PA0 5. Health, T. D., et al, Biochem Biophys Acta (1981) 640: 66. PA0 6. Martin, et al, Biochemistry (1981) 26: 4229. PA0 7. Hupfer, B., et al, Makromol Chem (1981) 182: 247. PA0 8. Regen, S. L., et al, J Am Chem Soc (1980) 102: 6640. PA0 9. Szoka, F. C., et al, Ann Rev Biophys Bioeng (1980) 9: 467. PA0 10. Szoka, F. C., et al, Proc Nat Acad Sci (USA) (1978) 75: 4194. PA0 11. Olson, F., et al, Biochem Biophys Acta (1979) 557: 9. PA0 12. Dancey, G. F., et al, J Immunol (1979) 255: 8015. PA0 13. Huang, A., et al, J Biol Chem (1980) 255: 8015. PA0 14. U.S. Pat. No. 4,483,929 for Liposomes With Glycolipid-linked Antibodies. PA0 15. Lowry, O. H., et al, J Biol Chem (1951) 193: 265.
3. Prior Art
A variety of therapeutic and diagnostic uses of liposomes have been reported. In many applications, liposomes are prepared to include surface-bound molecules such as small haptens, enzymes, antibodies, and other protein and non-protein molecules capable of conferring selected enzymatic or surface-recognition features to the liposomes. The surface molecules may function, in therapeutic applications, to target drug-containing liposomes to specific tissue or organ receptors (reference 1). In diagnostic applications of liposomes, the surface-bound molecules typically are ligands capable of binding with high affinity to analyte-related anti-ligand molecules, which may be either free in the assay reaction medium or carried on a solid surface. Binding between ligand and anti-ligand molecules leads to one of a variety of surface-attachment, agglutination, reporter-modulation or liposome-lysis events used in determining the presence and/or concentration of analyte in the reaction medium.
In some specialized uses, the surface-bound molecules may be lipid or lipid-like antigens, such as cardiolipin or glycolipid, which can be incorporated directly into the liposomes as part of the lipid components used in forming liposomes. In the more typical case, the molecules are relatively water-soluble antigens or proteins, which are preferably covalently attached to surface lipid components in the liposomes.
Several methods for coupling soluble molecules, such as proteins, to lipid structures, such as liposomes, have been reported. Some of these methods involve direct coupling of proteins to unmodified surface lipids by water-soluble cross-linking agents such as 1-ethyl,3-(3-dimethylaminopropyl)carbodiimide(EDCI) (reference 2), glutaraldehyde (reference 3), or suberimidate (reference 4). Cross-linking methods of this type have not been entirely satisfactory in that significant cross-linking of vesicles, or proteins or both may occur. The extent of specific ligand binding achievable is also generally quite low.
One protein coupling technique which allows relatively high levels of protein binding to liposomes has been described by Heath, et al (reference 5). The method involves periodate oxidation of glycosphingolipids in the liposome outer membranes, to form reactive surface aldehyde groups. Proteins are then attached to the aldehyde groups through Schiff-base formation, followed by reduction with NaBH.sub.4 or reductive amination with NaBH.sub.3 CN. Under optimal conditions, up to about 20% of the protein may be coupled to the oxidized vesicles, and coupling ratios between 100-200 .mu.g protein (IgG) per .mu.mole lipid may be achieved. One limitation of the method is the requirement for glycosphingolipids in the liposomes. General oxidative damage to liposomes caused by periodiate oxidation, and the need to remove periodate before protein coupling is carried out, are other limitations.
A second protein coupling method which has been shown to produce high coupling ratios is disclosed in U.S. Pat. No. 4,429,008 to Martin, et al. The liposomes in this method are formed by incorporating a thiol-reactive lipid, such as (N-[4-(p-maleimidophenyl)butyryl]phosphatidylethanolamine (MPB-PE) or (N-[3-(pyridyl-2-dithiopropionyl]phosphatidylethanolamine) (PDP-PE). The thiol-reactive liposomes are reacted with a protein bearing a free sulfhydryl group, or with one which has been thiolated to produce such a group, under conditions which lead to disulfide or thioether coupling to the thiol-reactive surface lipid. Coupling efficiencies of between 15%-30%, and coupling ratios greater than about 200 .mu.g protein (IgG)/.mu.mole lipid have been obtained (reference 6). The method is limited to proteins which have an available free sulfhydryl group or which can be thiolated without loss of protein activity.